We aim to continue our mammalian-cell studies that mechanistically characterize the pioneer round of translation and nonsense-mediated mRNA decay (NMD). We have found that pioneer translation initiation complexes consist of newly synthesized mRNAs that are bound at their 5' ends by the cap-binding protein (CBP) heterodimer CBP80-CBP20 and, if the mRNAs underwent splicing, at least one post-splicing exon-junction complex (EJC). Mammalian-cell mRNPs are targeted for NMD if they are bound by CBP80-CBP20, contain one or more EJCs that stably associate with some of the UPF NMD factors, and terminate translation in a way that triggers NMD. Steady-state translation initiation complexes, which we have shown derive from pioneer translation initiation complexes through translation-dependent and translation- independent mRNP remodeling steps, are bound at their 5' caps by eukaryotic translation initiation factor 4E (eIF4E) and lack detectable EJCs. Thus, steady-state translation initiation complexes, which support the bulk of cellular protein synthesis, are immune to NMD. While NMD is an important quality-control mechanism that typifies all eukaryotic cells, the restriction of NMD to newly synthesized mRNAs appears to be unique to mammalian cells. In AIM 1, we will continue to study the structure and molecular rearrangements of mRNPs before, during and after the pioneer round of translation. In AIM 2, we will further our collaboration with Rob Singer's lab to localize the cellular site of nucleus-associated NMD and, for comparison, the cellular site of cytoplasmic NMD using the fluorescent in situ hybridization of single RNA molecules. We will also use bimolecular fluorescence complementation coupled to Fvrster resonance energy transfer to localize within mammalian cells where particular trimolecular protein interactions that typify the pioneer round of translation and/or NMD occur. In AIM 3, we will use methods that we are developing to identify which EJCs are functional during NMD. Notably, while EJC function depends on EJC position within the NMD target, data indicate that not all exon-exon junctions are associated with an EJC and there can be compositional and/or functional differences among different EJCs. We will examine both of these issues. Through each of the three aims, using tools and technologies that we have established over the past three decades, we expect to continue making significant advances toward understanding the mechanism of NMD in mammalian cells and defining protein-protein and protein-mRNA interactions that typify mammalian-cell mRNPs.